Analysis of aroma and polyphenolic compounds in Saperavi red wine vinified in Qvevri

Abstract The purpose of this study is to analyze and characterize a Georgian red wine from Saperavi grape, obtained in Qvevri (Georgian traditional winemaking method), by using innovative techniques for the determination of the polyphenolic content, aroma, and its correlation to the sensory characteristics. This peculiar red wine, after high‐performance liquid chromatography with diode‐array detection and mass spectrometry (HPLC‐DAD‐MS), headspace solid‐phase microextraction–gas chromatography–mass spectrometry (HS‐SPME‐GC‐MS), and HS‐SPME‐GCxGC‐MS/TOF (two‐dimensional gas chromatography) chemical characterization showed a high polyphenol content (19.6 × 102 mg/L, 38.4% anthocyanins) and a wide range of volatile compounds, among which terpenes were associated with the aroma of flowers, lemongrass, and wood. Analyses were also conducted to determine the total polyphenol content correlated to antioxidant activity with the Folin–Ciocalteu spectrophotometric in vitro method (4.650 g GAE/L). In conclusion, for the first time on Saperavi wine, innovative techniques such as HPLC‐DAD‐MS, GC‐MS, and GCxGC‐MS/TOF were simultaneously applied in association with the traditional analytic techniques to perform a complete chemical characterization. These activities are part of a project about circular viticulture in the Georgian territory that will lead the production of traced quality wines and the valorization of the Georgian wine sector.


| INTRODUC TI ON
The ancient Georgian Qvevri traditional winemaking method is considered one of the country's cultural achievements and treasures in the UNESCO ICH list, and the Georgian wine is gradually acquiring its own identity (inscribed in 2013 "8.COM" on the Representative List of the Intangible Cultural Heritage of Humanity). "Qvevri" is an oval earthenware vessel used for the Georgian traditional winemaking procedures, which include fermentation and aging of wine. (Barisashvili, 2011). Qvevri is made of a type of clay whose manufacture is made by families of craftsmen according to the traditional technology diversified from region to region. Qvevri is buried in the earth, which guarantees an optimal temperature for the aging and conservation of the wine, and its egg-like shape | 6493 F. et al.
helps internal processes. The basic technological process, not uniquely identified, consists of pressing the grapes; then pouring juice, grape skins, stalks, and seeds into Qvevri; and then sealing and burying it in the ground for the fermentation process. The mixture thus obtained fills Qvevri for about 80%-85%, and the whole is stirred several times a day throughout the fermentation period. When fermentation is over, Qvevri is sealed and then left to age for 5-6 months (Barisashvili, 2011;Jackson, 2008;). Now, about 530 different grape varieties are registered in Georgia's ten wine regions, some of which are widespread. Most of them are however not cultivated, mainly present in collections or in experimental vineyards. The region with the most varieties is Kakheti, where 80 different varieties are registered. Kakheti is particularly notable for its several types of highest quality wines among other Georgian regions, and especially Kakhetian wine, traditionally produced in Qvevri. The highest quality Kakhetian wine is produced in specific micro-zones of Kakheti from grapes of Rkatsiteli, Saperavi, Kakhuri Mtsvane, Khikhvi, Kisi, and Kakhuri Mtsvivani (Glonti, 2010).
The quality of a wine and consequently the valorization of the production area depend on its numerous chemical components, whose presence or absence and amounts play an important role.
Wine aromatic profile plays a fundamental role in consumer preferences, that is, the result of the complex volatile fraction, formed by hundreds of compounds. During the aging process, wines undergo physicochemical transformations that can modulate color stability and spontaneous clarification and lead to a more complex flavor (Arfelli et al., 2007). The aroma of a wine depends on the simultaneous perception of a high number of volatile compounds, and the coupling of sensory analysis to GC-MS analysis can provide useful indications for an adequate evaluation of the aroma. Phenolic compounds, particularly abundant in wine, represent a further contribution to the sensory and chemical quality of the final product (Baiano et al., 2014); moreover, they can produce beneficial effects on human health (Watkins Ton, 1997).

HPLC-DAD-MS is the main technique suitable for identification
and quantification of phenolic compounds in food and wines.
Color is one of the main characteristics involved in the evaluation of appearance and therefore in the construction of the concept of quality by consumers, providing information about the type of wine, winemaking, and aging processes. Color can often led to the perception of other sensory characteristics as it allows one to anticipate the taste and/or olfactory properties according to the previous experience of the consumer (De Simón et al., 2008). This explains the importance of wine color in the acceptability of products (Morrot et al., 2001). The quali-quantitative determination of the wine polyphenolic content is also useful to do reliable considerations about specific biological properties such as antioxidant, anti-inflammatory, cardiovascular protection, and anti-neoplastic activities, mainly correlated with the presence of polyphenols (Baur & Sinclair, 2006;Calabriso et al., 2015;Dai & Mumper, 2010;Garcia-Alonso et al., 2009;Giovinazzo & Grieco, 2015;Paixao et al., 2007;Pandey & Rizvi, 2009).
Despite the wide availability of characterization studies regarding wines obtained from the most widespread cultivars, and despite the recognition of the quality and cultural tradition of the Saperavi grape and winemaking technique described before, at the authors' knowledge, there are few scientific studies in the literature with an in-depth characterization of the aromatic and polyphenolic profile of Saperavi wines.
According to the currently available results, Saperavi wines seem to have polyphenolic contents, in particular anthocyanins, comparable with more famous and studied red wines with high contents of polyphenols and anthocyanins such as Cabernet, Merlot, and Pinot Noir (Gil et al., 2012;Kekelidze et al., 2018;Kharadze et al., 2018;Mazza et al., 1999;Sergazy et al., 2019;Shalashvili et al., 2012;Wallace, 2011), whereas no data are available to evaluate its polyphenolic content and the aromatic and volatile fraction at the same time.
To the authors' knowledge, for the first time, on Saperavi wine, techniques such as HPLC-DAD-MS, GC-MS, and HS-SPME GCxGC-MS/TOF were simultaneously applied in association with the traditional analytic techniques to perform a complete chemical characterization. In addition, innovative headspace solid-phase microextraction, followed by comprehensive two-dimensional gas chromatography (HS-SPME-GCxGC-TOF), was adopted for the first time, providing a volatile fingerprint of Saperavi wine.

| Sample
The Saperavi BATONO wine was produced in 2014 and bottled in 2015 in a winery located in Kakheti (Georgia) using the "Qvevri" vinification technique. Physicochemical parameters of the Saperavi red wine are reported in Table 1. The general Qvevri vinification method is described in the "Introduction" section, in particular the cellar of Batono company is equipped with modern machinery and devices and wine aging is carried out in earthenware vessels, based upon enologist's decision, as described in the company's website. The analyses were carried out in triplicate on three different bottles of the same harvest year. The bottles were from the same batch, and the traceability of the samples is guaranteed by the traceability of the winery's quality production system.

| Analysis of volatile organic compounds
Volatile organic compounds (VOCs) were analyzed by both HS-SPME (solid-phase microextraction)-GC-MS and HS-SPME-GC × GC-TOF analyses. Initially, some tests were carried out to optimize the quantity of sample and the exposure temperature and time, in particular varying sample dilution (2.5-5 times), absorption temperature (30℃-60℃), and absorption time (10-30 min) to check any significant influence these changes could cause to the profile. For both the analyses, SPME conditions were set as follows, according to Domizio et al., 2018: 1 ml of wine was placed into a 20-mL screw cap vial fitted with PTFE/silicone septa, together with 2 g of NaCl, 4 ml of deionized water, and 40 μL of internal standard (ISTD) (ISTD: ethylacetate-D 8 ; 1-butanol-D 10 ; ethyl hexanoate-D 11 ; 5-methyl-hexanol; acetic acid-D 3 ; hexanoic acid-D 11 ; 3,4-dimethylphenol). The internal standard was used to normalize the analyte responses on the IS area, to minimize the instrumental error during the analysis time. After 5 min of equilibration, VOCs were absorbed exposing a 1-cm divinylbenzene/carboxen/polydimethylsiloxane SPME fiber (DVB/CAR/ PDMS by Supelco) at 60℃ for 10 min into the vial headspace under orbital shaking at 500 rpm and then immediately desorbed at 280℃ in a gas chromatograph injection port. Consistent SPME extraction conditions were ensured by a Gerstel MPS2 XL autosampler, equipped with a temperature-controlled agitated tray (Gerstel, Mülheim an der Ruhr, Germany). Samples were analyzed in triplicate.

| HS-SPME-GC-MS analysis
The VOCs absorbed, as described in the previous paragraph, were immediately desorbed at 280℃ in the injection port of a 7890a GC system (Agilent Technologies, Santa Clara, CA, USA) operating in a splitless mode, separated by a DB InnoWAX column (0.4 μm df × 0.2 mm i.d., 50 m) and detected by a quadrupole mass spectrometer 5975c MSD (Agilent Technologies, Palo Alto, CA, USA) operating in EI mode at 70 eV. Initial oven temperature was set at 40℃, held for 0.5 min, then raised to 260℃ at 6℃/min, and to finish held at 260℃ for 1 min. The helium carrier gas was set at a flow rate of 1.2 ml/min. The mass spectrometer worked in the mass range 29-350 m/z, with an electron ionization of 70 eV, and the total ion current chromatograms were recorded. Compounds were tentatively identified by comparing the mass spectra of each peak with those reported in the NIST11/NISTMass Spectral Library mass spectral database, with a minimum match factor of 80%. Peak identification was then confirmed by comparing their retention index, calculated by the generalized equation (Van Den Dool & Kratz, 1963) after injecting a mixture of linear alkanes (C 10 -C 26 ) in hexane in the same condition already described for sample analysis, with the literature (Chemistry WebBook). For many compounds, a positive identification was made by injecting authentic standards under the same analytical conditions. The peak areas relating to the tentatively identified compounds were normalized from Q (quantitation)-ion, and opportune internal standard (IS), according to their chemical properties, elution order, or both. The selection of the most suitable internal standard for each analyte was done, as described by Domizio et al., 2018.

| HS-SPME-GCxGC-TOFMS analysis
GCxGC was performed by a Compounds were tentatively identified comparing mass spectra with those reported in mass spectral NIST11/NISTMass database; identification was confirmed by their retention index as described in 1D analysis. were considered. Calibration was performed at the wavelength of the maximum UV-vis absorbance, by applying the correction of molecular weights. In particular, the anthocyanosidic compounds were calibrated at 520 nm with malvidin 3-glucoside (oenin); myricetin derivatives were calibrated at 350 nm with myricetin; quercetin and its derivatives were calibrated at 350 nm with quercetin 3-glucoside;

| HPLC-DAD-MS/TOF analysis
p-coumaric acid was calibrated at 308 nm with p-coumaric acid; hydroxycynnamic derivatives were calibrated at 330 nm with caffeic acid; vanillic acid was calibrated at 260 nm with vanillic acid; syringic acid was calibrated at 270 nm with syringic acid; resveratrol derivatives were calibrated at 308 nm with trans-resveratrol; gallic acid, catechin, epicatechin, procyanidins, and vanillin were calibrated at 280 nm, respectively, with gallic acid, catechin hydrate, and vanillin.
The determinations of the polyphenol contents were carried out in triplicate; the results are given as means, and the standard error was <5%.

| Antiradical activity
The antiradical activity was evaluated by using the stable radical

| Antioxidant activity with the Folin-Ciocalteu test
The antioxidant activity with the Folin-Ciocalteu spectrophotometric in vitro test was evaluated by using the procedure described in (Campo et al., 2016) with slight modifications. In particular, the absorbance at 725 nm was measured for a solution of the sample and the Folin-Ciocalteu reagent, after adding 20% Na 2 CO 3 and incubating for 40 min, using a calibration curve built by measuring the absorbance of five reaction solutions containing gallic acid at different concentrations. The phenol content of the sample is expressed as GAEs (gallic acid equivalents), as [g/L of sample)].

| RE SULTS AND D ISCUSS I ON
Wine is a complex alcoholic beverage containing volatile and nonvolatile components capable of interacting with aroma compounds to affect their volatility and concentration in the wine headspace and ultimately modify aroma perception and quality (Villamor & Ross, 2013). To profile and quantify volatile compounds, these can be extracted from wines using various techniques. The solidphase microextraction (SPME) technique is currently one of the most commonly used (Castro et al., 2008). The SPME technique is currently used to analyze volatile compounds from beverages and food (Calamai et al., 2012). According to the scientific literature,
The HS-SPME-GC-MS chromatogram of Saperavi wine showed a wide number of VOCs. Alcohols, esters, acids, aldehydes, and terpenes were determined. Terpenes are primary volatile compounds, called varietals. During the overripening of the grapes and during the aging of the wine, the terpenes undergo different chemical transformations which determine their decrease (Wilson et al., 1984).
Terpenes are present in very small concentrations, yet they have a considerable impact on the organoleptic properties of wine (Table 2).
Within this vast class of volatile components, monoterpene alcohols are those with the greatest sensory impact. In particular, linalool and geraniol are characterized by remarkably low perception thresholds. In this wine, linalool, α-terpineol, β-citronellol, α-terpinene, γterpinene, p-cymene, and terpinolene were found (Table 3). Grapes contribute to wine aroma with numerous compounds; however, it is during fermentation that the largest number of aroma compounds are formed, mainly alcohols, acids, and esters (Schreier, 1979). Some authors attribute the basic aroma of wine to four esters (ethyl acetate, isoamyl acetate, ethyl hexanoate, and octanoate) and two alcohols, (isobutyl and isoamyl alcohol or 3-methyl-1-butanol), all of which are fermentation products (Ferreira et al., 1995;Rapp & Mandery, 1986). Diethyl succinate or butanedioic acid diethyl ester was the major ester in Saperavi wine, associated with tropical fruit and floral descriptors. Isoamyl alcohol and 2-phenyl ethanol, with whiskey, malt, and honey, rose flavor, respectively, were the major higher alcohols found in this wine. analyzed under the same conditions, by comparison of the retention indices as retention linear indices (RLIs) with those from literature data and by the comparison of MS fragmentation patterns (MSs) with those reported in NIST11/NIST Mass Spectral Library mass spectral database, with a minimum match factor of 80%.
HS-SPME-GC × GC-TOF-MS analysis of the complex volatile fraction of Saperavi wine was submitted to advanced fingerprinting analysis of 2D chromatographic data (Figure 1). The use of HS-SPME-GC × GC-MS analysis permitted the creation of a comprehensive template matching fingerprinting, as shown in Figure 1. This method considers, as comparative aspect, each individual 2D peak together with its MS fragmentation pattern and time coordinates and includes them in a sample template created by the analyst that can be used to directly compare plots from different samples.
Each 2D peak corresponds to a single volatile compound. The deriving from SPME fiber bleeding and/or other molecules in monodimensional chromatography (34.00, 0.305 min. and 47.00, 075 min respectively). Aldehydes as nonanal, decanal, and benzeneacetaldehyde were present also in 1D chromatogram, but due to their low intensity, they were more evident in the 2D chromatogram ( Figure 1).
Red wine has a particularly high content of phenolic compounds with different structures. Flavonoids are the main compound, and the main flavonoid subclasses found in wine are flavan-3-ol monomers (catechin and epicatechin), oligomers and polymers (proanthocyanidins or condensed tannins), anthocyanins (malvidin derivatives in particular), and flavonols (quercetin, myricetin, and kaempferol and their glycosides). Also nonflavonoid compounds are present, such as hydroxybenzoic and hydroxycinnamic acids, phenolic alcohols, stilbenes, and ellagitannins. Anthocyanins are the main phenolic compounds of red wine, whose consumption has been partially related to the "French paradox." According to epidemiological studies, an increased consumption of anthocyanins can lower the risk of cardiovascular disease, the most common cause of mortality among men and women (Wallace, 2011). The presence of polyphenolic compounds in general is correlated with specific biological properties such as antioxidant, anti-inflammatory, cardiovascular protection, and anti-neoplastic activities (Baur & Sinclair 2006;Calabriso et al., 2015;Dai & Mumper 2010;Garcia-Alonso et al., 2009;Giovinazzo & Grieco, 2015;Paixao et al., 2007;Pandey & Rizvi 2009).
Malvidin 3-glucoside was found as the predominant anthocyanin ((3.4 ± 0.2) × 10 2 mg/L), but also the mono-glucosides of delphinidin, petunidin, and peonidin were present in high amounts, followed by acetyl and coumaroyl glucosides of malvidin. Quercetin was found F I G U R E 1 Comprehensive two-dimensional chromatography-mass spectrometry (GC×GC-TOF) color diagram and comprehensive template matching fingerprinting with the key identified volatile compounds of Saperavi wine only as its glucoside and glucuronide derivatives, in a total amount of 22 ± 1 mg/L; myricetin glucoside was also found in good quantity, and kaempferol was present only in traces. High amounts of caftaric and coutaric acids and esters, respectively, of caffeic and coumaric acids with tartaric acid were present (tot (275 ± 2) × 10 2 ), as well as gallic acid ((2.7 ± 0.1) × 10 2 ). The total of flavan-3-ol derivatives, including catechin and epicatechin oligomers and polymers (procyanidins), was (4.9 ± 0.2) × 10 2 mg/L expressed as catechin.